著者らは、プライマリRac1のFLOX / FLOXマウス胚線維芽細胞の彼の贈り物のためにトロント、オンタリオ州マウントサイナイ病院のDr。Rizaldyスコットに感謝します。この作品は、健康研究（CIHR、MOP 2009から93526）と自然科学とカナダの工学研究審議会（NSERC、RG 327372〜06）のカナダの協会からニュージャージー州まで営業の補助金によって支えられている。 SHとMWは、それぞれ、CIHRとNSERCからCGSMAとCGS - Mの賞でサポートされています。 スティーブハーレイとメラニーのウィルズは、この論文にも同様に貢献した。

融解細胞<ol><li>液体窒素から凍結マウス胚性繊維芽細胞（MEF）を（個々の胚初代培養から、以前に生成される）を取得。 </li><li> 37バイアルを浸漬し融解のセル内容が完全に融解するまで渦巻く° Cの水。 </li><li> 10cmの細胞培養プレートに10％ウシ胎児血清および1％ペニシリン - ストレプトマイシンを補充されたDMEMの9mLを追加。 </li><li>プレートに滴下融解した細胞懸濁液を追加し、37℃、5％CO 2インキュベーターで一晩細胞と場所のプレートを分散させるために軽くプレートを揺らし。プレート（約4時間後メッキ）に付着した細胞は、DMSO（凍結プロセスから）に敏感な場合、初期メディアが、細胞の後に新鮮な培地と交換できることに注意してください。また、凍結した細胞の数は、凍結後の細胞の回収率、および細胞の増殖速度に依存して解凍後の細胞のその合流点に注意してください。 </li></ol>継代細胞<ol start="5"><li>細胞がコンフルエントに本質的に100％（例：次の日）に成長しているときに、メディアをオフに吸引し、滅菌PBS 5mlの細胞を洗浄する。 </li><li> PBSを削除し、細胞がプレートからデタッチするまで、約5分間インキュベーターに10mMのEDTAと場所のプレートを含むトリプシン液1mLを加える。 </li><li>細胞がはがれるしたら、、塊を分散メディアの9mLを含む新しいプレート上に滴下この懸濁液1mLを追加し、37℃インキュベーターで一晩にこれを置くためにトリプシン処理細胞にメディアの9mLを加え、ピペット2-3回。メディアのFBSの存在が交互にトリプシンを不活性化する、細胞をトリプシンを希釈する最初の洗浄することができます。 </li></ol>カバースリップ上に細胞をカウントし、めっき<ol start="8"><li>インキュベーターから細胞を除去する、この時間を除いてバックの代わりに9mLのトリプシン処理細胞に5 - 6mLメディアを追加し、以前と同様に洗浄し、トリプシン処理。 </li><li>ピペットは、細胞の単一細胞の懸濁液中に完全な解離を確実に、そして別のマイクロチューブにトリパンブルーの15μLと組み合わせること15μL削除する。 </li><li>数回上下にピペッティングして、このセル/トリパンブルーサスペンションを混合し、血球計算盤に15μL加える。 </li><li>顕微鏡下で、死細胞は青色表示されます。血球計算板で4X4のグリッドのいずれかで、明確な/無色（生きた）細胞の数を数えます。他の3つの4X4のグリッド（A、B、CとDと表される）で、​​このカウントを繰り返して、サスペンションのμLあたりのセル数を求めるには、以下の計算式を使用します。 <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="式（1）" /></li><li>プロトコルの最適化を通じ、我々は、実験の終了時に可視化のために適切な細胞密度に30,000細胞の結果をメッキすることを決定した。 </li><li> 30,000細胞に必要な細胞懸濁液の体積を計算するには、次の式を使用します。 <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="式（2）" /></li><li>現在6つのウェルディッシュの各ウェルに滅菌ガラスカバースリップを配置し、各ウェルにメディアの2mLのを追加して、細胞懸濁液30,000細胞に必要な量から各ウェルに滴下追加。我々は、後の時点で蛍光顕微鏡を用いて細胞を可視化されるようにガラスのカバースリップがこの実験のために使用されることに注意してくださいではないすべてのアプリケーションは、必ずしも顕微鏡を必要とし、したがって、伝票が必要とされていない可能性がありますカバーします。 </li></ol>ウイルスの形質導入<ol start="15"><li>数時間または一晩（細胞はカバースリップに従っていることを保証するために十分な時間）の後、メディアを取り出し、無血清DMEM 1mLの細胞を洗浄する。 </li><li>ウイルスの追加のために各ウェルに無血清培地1mLのを追加。この実験では、アデノウイルスのCreはベクトルBiolabs社から商業的に入手した。 </li><li>以前のプロトコルの最適化は、500の感染多重度が（MOI）細胞の生存にほとんど、あるいはまったく効果が主のMEFに非常に効率的な遺伝子導入を提供することが示されている。 MOIは次のように計算することができます。 <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="式（3）" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="式（4）" /></li><li>感染している各ウェルに直接ウイルスの計算量を追加して、ゆっくりと一晩37℃のインキュベーター中にウイルスや場所の細胞を分散させるためにプレートを揺らし。 </li><li>翌朝は、ウイルスを含む細胞から培地を除去し、1mLの滅菌PBSで細胞を洗浄し、各ウェルに定期的なメディアの2mLの追加。 </li><li>この実験において例示Rac1の細胞の場合には、形態学的変化は、約72時間後に伝達における細胞内で発生した。 </li></ol>代表的な結果視覚化された細胞アクチン染料ファロイジン（イメージングのためだけに）で染色し、ゲルフ大学の高度な分析センターでライカTCS - SP5多光子共焦点レーザー走査型顕微鏡を用いて画像によるd。図1は、ウイルスにさらされていない同じ細胞と比較して、アデノのCreのウイルスにさらされたRac1のFLOX / FLOX細胞の収縮と伸長形態を示しています。 <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="図1" /> 図1と同じソース（右）から非感染細胞と比較してアデノウイルスのCre（左）に感染してRac1のFLOX / FLOXセル間の比較。

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

動物実験は動物のケアに関するカナダの理事会が定めるガイドラインおよび規則に従って行った。

付属のビデオでは、組換えウイルスはCreリコンビナーゼを用いた in vitro における消費税の特定の遺伝子にどのように使用できるかを例示している。このプロトコルの開発は、しかし、アドレッシング価値があるいくつかの特定の点を明らかにしました。 <ul><li>アデノウイルスを使用する際、適切な安全手順を常に使用すべきである。ウイルスでの作業中に白衣と手袋を常に着用してください。メディアとプラスチック容器は、少なくとも30分間、10％の漂白剤で治療されるべきである、とプラスチック製の食器は、その後オートクレーブしてください。 </li><li>これはウイルスの力価に影響を与えるため、導入効率を減らすことができるように繰り返されたウイルス株の凍結融解は避けてください。ウイルスストックの小さなボリュームは別々のチューブに等分されるべきである。 </li><li> 50から500に至るまでMOISは、メソッドの開発中にテストされたと効率的な伝達率は、細胞の生存に悪影響することなく、ほとんどのケースで観察された。 </li><li>アデノ- GFPは、迅速形質導入効率をスクリーニングする方法の開発中に使用されていました。このコントロール（または空のベクター）は、細胞の変化が発生しないだけで、ウイルス感染を確保するため、実験中に実施されるべきである。同様に、あなたの細胞への関心のfloxed遺伝子を切り出すことはあらゆる細胞の変化（形態や生存率、すなわち変化）が発生しない場合は、アデノ- GFPと同じ細胞型を感染させると、そのウイルスの伝達を示すために使用することが重要コントロールですです発生。 </li></ul>文化のシグナル伝達カスケードをの調査を超えて、アデノのCreのアプローチはまた、条件付きで実験動物3,4からの遺伝子を除去するため、および生物6内の細胞の特定の集団を標識するためにin vivoで採用されている。アデノウイルスシステムはまた、宿主細胞にCreリコンビナーゼ以外の遺伝子を導入するために適応させることができます。この送達ベクターを使用しての二つの主要な利点はその高い伝達と遺伝子デリバリー効率、及び宿主DNAとの統合の欠如です。しかし、新規な組換えアデノウイルスの発生は、労働集約することができます。プロトコルは、したがって、ここで説明する以前に開発されたアデノのCreのウイルスを利用する、と関心、Rac1の私​​たちのfloxed対立遺伝子とのことをカップリングさせることによりアデノウイルスシステムのパワーを利用。実験からこの条件ノックを通じ、我々はfloxed Rac1の対立遺伝子を運ぶMEFのアデノのCreのウイルスを使用して、その伝達を確認していることは確かにRac1の切除は、Rac1の欠損細胞の細胞形態の特徴2,5の変化につながる可能性がありません。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ad-CMV-Cre virus</td>
<td>&nbsp;</td>
<td>Vector Biolabs</td>
<td>1045</td>
<td>&nbsp;</td>
<tr>
<td>DME High glucose media 500mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3002201</td>
<td>&nbsp;</td>
<tr>
<td>FBS Canadian origin 500mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CAA15-701</td>
<td>&nbsp;</td>
<tr>
<td>PEN-STREP 1X solution 100mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>Texas Red-X phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T7471</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4549</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

遺伝的に様々な細胞のシグナル伝達カスケードの特定のコンポーネントを削除する機能は、近代的なシグナル伝達の解析に不可欠なツールとなっています。この条件の削除を実現する一つの特定のメソッドでは、Cre - loxP組システムの使用を介してです。このメソッドは、Creリコンビナーゼのタンパク質に特異的な認識配列であるloxP部位、で目的の遺伝子に隣接伴います。 Creを介した組換えloxP部位でのイベント、および介在する遺伝子3のその後の切除でこの酵素の結果に、いわゆるfloxed（loxP部位によって挟まれた）DNAの露出。いくつかの異なる方法は、興味のあるサイトにCreリコンビナーゼを管理するために存在します。このビデオでは、我々はfloxed Rac1の対立遺伝子1を含む胚から得られた一次マウス胚性繊維芽細胞（MEF）を感染させるためにCreリコンビナーゼ遺伝子を含むアデノウイルスの使用を示します。私たちの実験のためにRac1のMEFのを選択するための論理的根拠は、その明確な形態学的変化が原因でアクチン細胞骨格の2,5の変化に、Rac1の削除時に見られるようです。ウイルスの形質導入とCreの発現後72時間は、細胞はアクチン染料ファロイジンを用いて染色し、共焦点レーザー走査顕微鏡を用いて画像化した。それは、アデノ- CreをウイルスにさらされていたMEFのは、非感染細胞と比較して以前の報告2,5と一致して契約し、形態に長い現われたことが観察された。 Creリコンビナーゼの配信のアデノウイルスの方法は、アデノのCreのウイルスが容易に利用できるように有利である、と細胞のほぼ100％にCreを経由して遺伝子の欠失は、最適化されたアデノウイルス感染を達成することができます。

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

このビデオでは、floxed Rac1の対立遺伝子を運ぶ主要なマウス胚性線維芽細胞を感染させるためにCreリコンビナーゼ遺伝子を有するアデノウイルスを使用してください。

培養一次マウス胚線維芽細胞におけるシグナル伝達分子のアデノウイルス媒介遺伝子除去

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

24.8K ビュー.  University of Guelph. このビデオでは、floxed Rac1の対立遺伝子を運ぶ主要なマウス胚性線維芽細胞を感染させるためにCreリコンビナーゼ遺伝子を有するアデノウイルスを使用してください。

ビデオ: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

生物学

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4つの転写因子とマウス胚線維芽細胞を再プログラムすることによって誘導多能性幹細胞の生成、ドキシサイクリンは、レンチウイルス伝達系の誘導性

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

胚脊髄交連軸索アンテナの向きに拡散性のシグナル伝達分子の能力を検定する

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

培養中の細胞小器官輸送<em&gt;ショウジョウバエ</em&gt;細胞：S2細胞ラインと一次ニューロン。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

インスリン刺激に対するグルコース取り込み測定と応答<em&gt;インビトロ</em&gt;培養ヒト初代筋管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...